臺灣博碩士論文加值系統

P血型系統是由醣脂質抗原組成的系統，然而其分子遺傳特性卻尚未釐清。其中又以缺乏P1, Pk and P三種抗原表現的p血型個體極為罕見，過去的研究認為是α (1, 4) galactosyltransferase (A4GALT)活性缺失造成Pk抗原無法合成所致。本研究第一個部份是探討一位臺灣p血型及其家族基因研究。首先我們以polymerase chain reaction和直接定序分析A4GALT 基因的coding region，結果於9位家族成員中共有2位具homozygous 核苷酸418和428之間呈現複合式剔除11 bp暨插入34 bp的突變現象，簡稱為418-428 (del-ins)。如此變異經由軟體預測會引發reading frame shift現象，而造成 premature stop codon。因此當我們以重組工程技術建構此突變型的construct並轉染於CHO細胞後，以flow cytometry與免疫螢光染色法僅見細胞微量表現Pk抗原。此外，我們進行Galβ1-4Glc-PA轉移為Galα1-4Galβ1-4Glc–PA的酵素活性試驗並以HPLC醣類分析技術去測試A4GALT酵素活性，結果p mutant僅出現微量的波峰。目前P血型系統的分子特性與決定P1/P2血型的主要調控基因仍是一大謎題，因此我們第二個研究部份是自A4GALT 基因的coding region多型性之間，尋找出調控P1抗原表現的相關因子。首先我們收集各100位血清型確認為P1與P2血型的捐血人並以PCR-RFLP法分析其A4GALT 基因109A>G、903C>G與987G>A 。依據初步的統計資訊中，我們自109A>G和 987G>A的表現頻率去估測其所形成的haplotype頻率可以有意義地去區分P1與P2血型二個族群。所以我們進一步地以PCR-SSP與直接定序去分析A4GALT haplotype 109與 987。經重新統計分析發現，結果haplotype GA與P1血型的表現具相關性(OR, 9.33; 95%CI, 4.31-20.19)，然而haplotype AG與AA傾向於P2血型的表現。因此我們以定點突變建構兩種haplotype AG與GA，並將帶有這兩種haplotype穩定性轉染的細胞株，以flow cytometry比較表面CD77的表現，結果發現haplotype GA僅略高於haplotype AG，因此目前仍不能完全證實haplotype GA形成 P1的表現型是因為有較高的A4GALT酵素活性才造成。

The P blood group system is the glycolipid antigen system for which the molecular genetic basis has not yet been fully illustrated. Individuals of p phenotype are very rare and lack P1, Pk and P, presumably as a result of deficiency in the enzyme α (1, 4) galactosyltransferase (A4GALT), that responsible for synthesis of Pk antigen. The aim of the study is to explore the molecular background of a family with p phenotype in Taiwan. The coding region of the A4GALT gene was analyzed by polymerase chain reaction and direct sequencing. Of the nine family individuals, the two with p phenotype are homozygous for the complex mutation with a combined deletion and insertion occurred between the nucleotide 418 and 428; 418-428(del-ins). This alteration result in a shift in the reading frame and a premature stop codon. Therefore, the minimal expression of Pk was showed by flow cytometry and immunofluorescent method on CHO cells transfected with the construct containing the complex mutation. Furthermore, carbohydrate analysis with HPLC was performed to analyze A4GALT activity by transfering Galβ1-4Glc-PA to Galα1-4Galβ1 -4Glc–PA. The peak was barely detected in CHO cells transfected with p mutant of A4GALT construct.
The molecular genetic basis of the P blood group system and the P1/P2 phenotypes determination is still unclear. The second aim of the study is to look for polymorphisms in the coding region of A4GALT gene responsible for P1 expression. We collected serologically confirmed P1+ (P1 phenotype; n=100) and P1- (P2 phenotype; n=100) blood donors and analyzed the polymorphisms 109A>G, 903G>C and 987G>A of A4GALT by the methods of polymerase chain reaction and restriction fragment length polymorphism. From the preliminary data, allele frequencies for 109A>G and 987G>A estimated haplotype frequencies differed significantly between P1and P2 groups. Then the haplotype of A4GALT 109 and 987 was further analyzed by the method of PCR-specific sequence primer and direct sequencing. The results showed the haplotype of GA was found to be P1-association (OR, 9.33; 95%CI, 4.31-20.19), however, AG and AA were P2-specific. Therefore, haplotype of AG and GA at nucleotide position of 109 and 987 sites of pcDNA 4/V5/A4GALT were generated by site directed mutagenesis. The stable clones of haplotypes AG and GA were selected to compare the A4GALT enzyme activity by flow cytometry to detect CD77 expression on cell surface. Nevertheless, the A4GALT activities in transfected cells with haplotype GA were only little higher than haplotype AG. Therefore, it is still not concluded haplotype GA with high enzyme activity for P1 expression.

中文摘要……………………………………………………………………….…….Ⅰ
英文摘要……………………………………………………………………………. Ⅱ
誌謝…………………………………………………………………………………..Ⅳ
目錄…………………………………………………………………………………..Ⅴ
表目錄……………………………………………………………...………………. .Ⅷ
圖目錄……………………………………………………………………………..…Ⅸ
附錄圖表目錄…………………………………………………………….……….…Ⅹ
縮寫簡索表……………………………………………..…………………….….... .ⅩI
儀器與藥品……………………………………………..…………………….…... ⅩⅡ
第一章 緒論…………………………………………………………………………1
一、P相關血型系統…………………………………………………….……..1
二、P相關血型系統的分子特性……………………………………………...1
三、p血型與anti-PP1Pk……………………………………………………….3
四、p血型與A4GALT基因變異的相關性……………………………….…..3
五、調控P1/P2 血型表現的P1基因…………………………………………4
第二章 研究動機……………………………………………………………………7
第三章 材料與方法…………………………………………………………………8
一、P血型鑑定………………………………………………………………...8
(一) 檢體來源………………………………………………………………8
(二) 血清學方法測定P血型系統表現……………………………………8
(1)紅血球凝集試驗試管法( tube method) ……………………………8
(2)管柱凝集法(column agglutination test) ………………………..…..9
二、A4GALT基因分析………………………………………………………....9
(一) Genomic DNA萃取……………………………………………………9
(二) A4GALT基因的序列分析…………………………………………….10
(1) A4GALT基因coding region序列(-140) 到 +555………………..10
(2) A4GALT基因coding region序列386到1120……………………12
(3)DNA定序…………………………………………………….…….13
(三) 利用PCR-RFLP(Restriction fragment length polymorphism)區分A4GALT基因多型性 ………………………………………….……13
(1) A4GALT 109A>G基因多型性……………………………….……13
(2) A4GALT 903C>G基因多型性……………………………….……14
(3) A4GALT 987G>A基因多型性…………………………………….15
(四) 利用SSP-PCR (Sequence-Specific Primer-Polymerase Chain Reaction)區分A4GALT基因多型性…………………………………………….16
三、質體重組工程 (Recombinant plasmid construction) ……………………...18
(一) 聚合酶連鎖反應 (PCR) ………………………………..……………18
(二) 純化PCR產物………………………………………………………..20
(三) 質體接合(ligation) ………………………………………………...…20
(四) 轉形(transformation)反應…………………………………………….22
(五) 小量質體萃取………………………………..……………………….22
(六) 大量質體萃取………………………………………………….……..23
(七) 定點突變………………………………………………….…….…….25
四、細胞培養與轉染…………………………………………………….…….26
(一) 繼代細胞培養與轉染………………………………………….……..26
(二)西方墨點法(Western blotting) ………………………………….……..29
(1) SDS-PAGE分析…………………………………………….……...29
(2)西方墨點轉漬法…………………………………………….………31
(3) PCR…………………………………………………………………34
五、A4GALT酵素活性分析…………………………………….…………….35
(一)流式細胞儀分析(Flow cytometry analysis) …………………………..36
(二)免疫螢光染色法(Immunofluorescent staining method) ………...........37
(三) Enzyme activity assay…………………………………........................38
(1) membrane fraction萃取…………………………………................38
(2) A4GALT酵素純化………………………………….......................39
(3) A4GALT酵素活性試驗……………………………………………40
(4)高效能液相層析儀系統(HPLC) ………………………………..…40
第四章 結果………………………………………………………………………...42
第五章 討論………………………………………………………………………...46
第六章 參考文獻…………………………………………………………………...51
表…………………………………………………………………………………….57
圖…………………………………………………………………………………….63
附錄………………………………………………………………………………….72

1.Daniels G. Human Blood Groups, 2nd edn. Oxford, Blackwell Science, 2002.
2.Daniels GL, Fletcher A, Garratty G, Henry S, Jørgensen J, Judd WJ, Levene C, Lomas-Francis C, Moulds JJ, Moulds JM, Moulds M, Overbeeke M,
Reid ME, Rouger P, Scott M, Sistonen P, Smart E, Tani Y, Wendel S, Zelinski T. Blood group terminology 2004: From the ISBT Committee on Terminology for Red Cell Surface Antigens. Vox Sang 2004; 87: 304–16.
3.von dem Borne AEGK, Bos MJE, Joustra-Maas N, TROMP I, V A N Wijngaardenb-Doius R, P A T. A murine monoclonal IgM antibody specific for blood group P antigen (globoside). Br J Haematol 1986; 63: 35–46.
4.Shevinsky LH, Knowles BB, Damjanov I, Solter D. Monoclonal antibody to murine embryos defines a stage-specific embryonic antigen expressed on mouse embryos and human teratocarcinoma cells. Cell 1982; 30: 697–705
5.Kelus A, Gurner BW, Coombs RRA. Blood group antigens on HeLa cells shown by mixed agglutination. Immunology 1959; 2: 262–7.
6.Bono R, Cartron JP, Mulet C, Avner P, Fellous M. Selective expression of blood group antigens on human teratocarcinoma cell lines. Rev Franc Transfus Immuno- Hémat 1981; 24: 97–107.
7.Nudelman E, Kannagi R, Hakomori S, Parsons M. A glycolipid antigen associated with Burkitt lymphoma defined by a monoclonal antibody. Science 1983; 220: 509–11.
8.Kasai K, Galton J, Terasaki PI, Wakisaka A, Kawahara M, Root T, Hakomori S I. Tissue distribution of the Pk antigen as determined by a monoclonal antibody. J Immunogenet 1985; 12:213–20.
9.Wiels J, Fellous M, Tursz T. Monoclonal antibody against a Burkitt lymphoma- associated antigen. Proc Natl Acad Sci USA 1981; 78: 6485–8.
10.Spitalnik PF, Spitalnik SL. The P blood group system: biochemical, serological, and clinical aspects. Tranfus Med Rev 1995; 9: 110–22.
11.Haataja S, Tikkanen K, Liukkonen J, Francois-Gerard C, J Finne. Characterization of a novel bacterial adhesionspecificity of Streptococcus suis recognizingblood group P receptor oligosaccharides. J Biol Chem 1993; 268: 4311–17.
12.Eder AF, Spitalnik SL. Blood group antigens as receptors for pathogens. In: A Blancher, J Klein, WW Socha, eds. Molecular Biology and Evolution of Blood Group and MHC Antigens in Primates. Berlin: Springer-Verlag 1997, 268–304.
13. Puri A, Hug P, Jernigan K, Barchi J, Kim H Y, Hamilton J, Wiels J, Murray G J, Brady RO, Blumenthal R.The neutral glycosphingolipid globotriaosyl- ceramide promotes fusion mediated by a CD4-dependent CXCR4-utilizing HIV type 1 envelope glycoprotein. Proc Natl Acad Sci USA 1998; 95: 14435–40.
14.Hammache D, Yahi N, Maresca M, Piéroni G, Fantini J. Human erythrocytes glycosphingolipids as alternative cofactors for human immunodeficiency virus type 1 (HIV-1) gp120 and reconstituted membrane microdomains of glycosphingolipids (Gb3 and GM3). J Virol 1999; 73: 5244–8.
15.Hellberg Å, Ringressi A, Yahalom V, Sa¨fwenberg J , Reid ME, Olsson ML. Genetic heterogeneity at the glycosyltransferase loci underlying the GLOB blood group system and collection. Br. J. Haematol. 2004; 125: 528–36.
16.Brown KE, Hibbs JR, Gallinella G, Anderson SM, Lehman ED, McCarthy P, Young NS. Resistance to parvovirus B19 infection due to lack of virus receptor (erythrocyte P antigen). N Engl J Med 1994; 330: 1192–6.
17.Levine P, Bobbitt OB, Waller RK, Kuhmichel A. Isoimmunization by a new blood factor in tumor cells. Proc Soc Exp Biol NY 1951; 77: 403–5.
18.Sanger R. An association between the P and Jay systems of blood groups. Nature 1955; 176: 1163–4.
19.Söderström T, Enskog A, Samuelsson BE, Cedergren B. Immunoglobulin subclass (IgG3) restriction of anti-P and anti-Pk antibodies in patients of the rare p blood group. J Immunol 1985; 134: 1–3.
20.Lindström K, dem Borne AEGK, Breimer ME, Cedergren B, Okubo Y, Rydberg L, Teneberg S, Samuelsson BE. Glycosphingolipid expression in spontaneously aborted fetuses and placenta from blood group p women: evidence for placenta being the primary target for anti-Tja-antibodies. Glycocon J 1992; 9: 325–9.
21.Rydberg L, Cedergren B, Breimer ME, Lindström K, Nyholm PG, Samuelsson BE. Serological and immunochemical characterization of anti-PP1Pk (anti-Tja) antibodies in blood group p individuals: blood group A Type 4 recognition due to internal binding. Mol Immunol 1992; 29: 1273–86.
22.Cantin G, Lyonnais J. Anti-PP1Pk and early abortion. Transfusion 1983; 23:350–1.
23.Levene C, Sela R, Rudolphson Y. Hemolytic disease of the newborn due to anti-PP1Pk (anti-Tja). Transfusion 1977; 17: 569–72.
24.Lin CK, Mak KH, Cheng CK, Yang CP. The first case of the p phenotype in a Ghurka Nepalese. Immunohematology 1998; 14: 30–2.
25. Levine P, Bobbitt OB, Waller RK, Kuhmichel A. Isoimmunization by a new blood factor in tumor cells. Proc Soc Exp Biol NY 1951; 77: 403–5.
26.Hellberg Å, Anne-Christine Schmidt-Melbye, Marion E. Reid, and Martin L. Olsson Expression of a novel missense mutation found in the A4GALT gene of Amish individuals with the p phenotype. Transfusion 2008; 48: 479–87.
27.Steffensen R, Carlier K, Wiels J, Leveryi SB, Stroud M, Cedergren B, Sojka B N, Bennett EP, Jersild C, Clausen H. Cloning and expression of the histo-blood group Pk UDP-galactose: Galbeta -4glcbetal-cer alphal, 4-galactosyltransferase. Molecular genetic basis of the p phenotype. J Biol Chem 2000; 275: 16723–9.
28.Kojima Y, Fukumoto S, Furukawa K, Okajima T, Wielsi J, Yokoyama K, Suzuki Y, Urano T, Ohta M, and Furukawa K Molecular cloning of globotriaosylceramide / CD77 Synthase, a Glycosyltransferase that initiates the Synthesis of Globo Series Glycosphingolipids. J Biol Chem 2000; 275: 15152–6.
29.Hellberg Å, Chester MA, Olsson ML. Two previously proposed P1 / P2-differentiating and nine novel polymorphisms at the A4GALT(PK) locus do not correlate with the presence of P1 blood group antigen. BMC Genet 2005; 6: 49–59.
30.Tilley L, Green C, Daniels G. Sequence variation in the 5´untranslated region of the human A4GALT gene is associated with, but does not define, the P1 blood-group polymorphism. Vox Sang 2006; 90: 183–8.
31.Furukawa K, Iwamura K, Uchikawa M, Sojka BN, Wiels J, Okajima T, Urano T, Furukawa K. Molecular Basis for the p Phenotype. J Biol Chem 2000; 275: 37752–6.
32.Yan L, Zhu F, Xu X, Zantek ND. Molecular basis for p blood group phenotype in China. Transfusion 2004; 44: 136–8.
33.Koda Y, Soejima M, Sato H, Maeda Y, Kimura H. Three-base deletion and one-base insertion of the α (1, 4) galactosyltranserase gene responsible for the p phenotype. Transfusion 2002; 42: 48–51.
34.Hellberg Å, Steffensen R, Yahalom V, Sojka BN, Heier HE, Levene C, Poole J, Olsson ML. Additional molecular bases of the clinically important p blood group phenotype. Transfusion 2003;43: 899–907.
35.Iwamura K, Furukawa K, Uchikawa M, Sojka BN, Kojima Y, Wiels J, Shiku H, Urano T, Furukawa K. The blood group P1 identical to the Gb3/CD77 synthase gene. A cule to the solution of the P1/P2/p puzzle. J Biol Chem 2003; 278: 44429–38.
36.Geoff Daniels.The molecular genetics of blood group polymorphism. Transplant Immunology 2005; 14: 143 –53.
37.Fletcher KS, Bremer EG, Schwarting GA. P blood group regulation of glycosphingolipid levels in human erythrocytes. J Biol Chem 1979; 254: 11196–11198.
38.Hase S, Ikenaka T, Matsushima Y. Analyses of Oligosaccharides by Tagging the Reducing End with a Fluorescent Compound. J Biochem. 1979; 85: 989-94.
39.Hase S, Hara S, Matsushima Y. Tagging of Sugars with a Fluorescent Compound, 2-Aminopyridine. J Biochem 1979; 85: 217–20.
40.Sugimoto I, Onimatsu H, Fujie M, Usami S, Yamada T. vAL-1, a novel polysaccharide lyase encoded by chlorovirus CVK2. FEBS Letters. 2004; 559: 51–6.
41.Takeuchi T, Hayama K, Hirabayashi J, Kasai K. Caenorhabditis elegans N-glycans containing a Gal-Fuc disaccharide unit linked to the innermost GlcNAc residue are recognized by C. elegans galectin LEC-6. Glycobiology 2008; 18: 882–90.
42.Oka S, Terayama K, Imiya K, Yamamoto S, Kondo A, Kato I, Kawasaki T. The N-glycan acceptor specificity of a glucuronyltransferase, GlcAT-P, associated with biosynthesis of the HNK-1 epitope. Glycoconjugate J 2000; 17: 877–85.
43.Takakura Y, Tsukamoto H, Yamamoto T. Molecular Cloning, Expression and Properties of an α/β-Galactoside α2, 3-Sialyltransferase from Vibrio sp. JT-FAJ-16. J Biochem 2007; 142: 403–12.
44.Terada M, Khoo KH, Inoue R, Chen CI, Yamada K, Sakaguchi H, Kadowaki N, Ma BY, Oka S, Kawasaki T, Kawasaki N. Characterization of Oligosaccharide Ligands Expressed on SW1116 Cells Recognized by Mannan-binding Protein. J Biol Chem 2005; 280: 10897–913.
45.Keusch JJ, Manzella SM, Nyame KA, Cummings RD, Baenziger JU. Cloning of Gb3 Synthase, the Key Enzyme in Globo-series Glycosphingolipid Synthesis, Predicts a Family of α1, 4- Glycosyltransferases Conserved in Plants, Insects, and Mammals. J Biol Chem 2000; 275: 25315–21.
46.Ju T, Cummings RD. A unique molecular chaperone Cosmc required for activity of the mammalian core 1 β3-galactosyltransferase. PNAS 2002; 99: 16613–8.